Starches such as corn, potato, wheat, manioc and rice starch are used as the starting material in commercial large scale production of sugars, such as high fructose syrup, high maltose syrup, maltodextrins, amylose, G4-G6 oligosaccharides and other carbohydrate products such as fat replacers.
Degradation of Starch
Starch usually consists of about 80% amylopectin and 20% amylose. Amylopectin is a branched polysaccharide in which linear chains .alpha.-1,4 D-glucose residues are joined by .alpha.-1,6 glucosidic linkages. Amylopectin is partially degraded by .alpha.-amylase, which hydrolyzes the 1,4-.alpha.-glucosidic linkages to produce branched and linear oligosaccharides. Prolonged degradation of amylopectin by .alpha.-amylase results in the formation of so-called .alpha.-limit dextrins which are not susceptible to further hydrolysis by the .alpha.-amylase. Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by a debranching enzyme. The remaining branched oligosaccharides can be depolymerized to D-glucose by glucoamylase, which hydrolyzes linear oligosaccharides into D-glucose.
Amylose is a linear polysaccharide built up of D-glucopyranose units linked together by .alpha.-1,4 glucosidic linkages. Amylose is degraded into shorter linear oligosaccharides by .alpha.-amylase, the linear oligosaccharides being depolymerized into D-glucose by glucoamylase.
In the case of converting starch into a sugar, the starch is depolymerized. The depolymerization process consists of a pretreatment step and two or three consecutive process steps, namely a liquefaction process, a saccharification process and, depending on the desired end product, optionally an isomerization process.
Pre-treatment of Native Starch
Native starch consists of microscopic granules which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. During this "gelatinization" process there is a dramatic increase in viscosity. As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or "liquefied"so that it can be handled. This reduction in viscosity is today mostly obtained by enzymatic degradation.
Liquefaction
During the liquefaction step, the long-chained starch is degraded into smaller branched and linear units (maltodextrins) by an .alpha.-amylase (e.g. Termamyl.TM., available from Novo Nordisk A/S, Denmark). The liquefaction process is typically carried out at about 105-110.degree. C. for about 5 to 10 minutes followed by about 1-2 hours at about 95.degree. C. The pH generally lies between about 5.5 and 6.2. In order to ensure an optimal enzyme stability under these conditions, calcium is added, e.g. 1 mM of calcium (40 ppm free calcium ions). After this treatment the liquefied starch will have a "dextrose equivalent" (DE) of 10-15.
Saccharification
After the liquefaction process the maltodextrins are converted into dextrose by addition of a glucoamylase (e.g. AMG.TM., available from Novo Nordisk A/S) and a debranching enzyme, such as an isoamylase (see e.g. U.S. Pat. No. 4,335,208) or a pullulanase (e.g. Promozyme.TM. available from Novo Nordisk A/S) (see U.S. Pat. No. 4,560,651). Before this step the pH is reduced to a value below 4.5, e.g. about 3.8, maintaining the high temperature (above 95.degree. C.) for a period of e.g. about 30 min. to inactivate the liquefying .alpha.-amylase to reduce the formation of short oligosaccharides called "panose precursors" which cannot be hydrolyzed properly by the debranching enzyme.
The temperature is then lowered to 60.degree. C., glucoamylase and debranching enzyme are added, and the saccharification process proceeds for about 24-72 hours.
Normally, when denaturing the .alpha.-amylase after the liquefaction step, a small amount of the product comprises panose precursurs which cannot be degraded by pullulanases or AMG. If active amylase from the liquefaction step is present during saccharification (i.e. no denaturing), this level can be as high as 1-2% or even higher, which is highly undesirable as it lowers the saccharification yield significantly. For this reason, it is also preferred that the .alpha.-amylase is one which is capable of degrading the starch molecules into long, branched oligosaccharides (such as, e.g., the Fungamyl.TM.-like .alpha.-amylases) rather than shorter branched oligosaccharides.
Isomerization
When the desired final sugar product is e.g. high fructose syrup, the dextrose syrup may be converted into fructose by enzymatic isomerization. After the saccharification process the pH is increased to a value in the range of 6-8, preferably about pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immobilized glucose isomerase (such as Sweetzyme.TM., available from Novo Nordisk A/S).
Debranching Enzymes
Debranching enzymes which can attack amylopectin are divided into two classes: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41), respectively. Isoamylase hydrolyses .alpha.-1,6-D-glucosidic branch linkages in amylopectin and .beta.-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by their limited action on .alpha.-limit dextrins.
When an acidic stabilised "Termamyl.TM.-like" .alpha.-amylase is used for the purpose of maintaining the amylase activity during the entire saccharification process (no inactivation), the degradation specificity should be taken into consideration. It is desirable in this regard to maintain the .alpha.-amylase activity throughout the saccharification process, since this allows a reduction in the amyloglucidase addition, which is economically beneficial and reduces the AMG.TM. condensation product isomaltose, thereby increasing the DE (dextrose equivalent) yield.
It will be apparent from the above discussion that the known starch conversion processes are performed in a series of steps, due to the different requirements of the various enzymes in terms of e.g. temperature and pH. It would therefore be desirable to be able to engineer one or more of these enzymes so that the overall process could be performed in a more economical and efficient manner. One possibility in this regard is to engineer the otherwise thermolabile debranching enzymes so as to render them more stable at higher temperatures. The present invention relates to such thermostable debranching enzymes, the use of which provides a number of important advantages which will be discussed in detail below. It also relates to starch debranching enzymes with an altered substrate specificity.